Glycogen Phosphorylase

=Introduction= Glycogen phosphorylase catalyzes the hydrolysis of glycogen to generate glucose-1-phosphate and shortened glycogen molecule and is considered the rate limiting step in the degradation of glycogen. It is a part of the glucosyltransferase family and acts on the α-1,4-glycosidic linkage; the phosphorylase comes to a standstill 4 residues from an α-1,6-branchpoint, where debranching enzyme takes over. The glucose-1-phophate is then further degraded via the pathway of glycolysis. Studies have found that mammals have liver, muscle and brain isoforms of phosphorylase but it is found among all species; muscle glycogen phosphorylase is present to degrade glycogen to forms of energy by means of glycolysis during muscle contractions and liver glycogen is present to regulate the blood glucose levels within the blood. =Structure and Function= Glycogen phosphorylase is a dimer consisting of two identical subunits and has an essential cofactor, pryridoxal phosphate (PLP). Glycogen phosphorylase can be found in two different states, glycogen phosphorylase a (GPa) and glycogen phosphorylase b (GPb) The difference in the structures is due to phosphorylation of the Ser-14 residue which results in the active form (GPa). Protein phosphatases dephosphorylate the GPa to the inactive form, also known as GPb. Both forms of glycogen phosphorylase can also be found in T and R states where T is the inactive state because it appears to have a low affinity for substrate and R is the active state where it appears to have a greater affinity for substrate.

The secondary structures of T and R states of GPa and b are similar with an N-terminal domain and a C-terminal domain .Each domain also contains subdomains which undergo conformational changes on the interconversion of T and R states. C terminal domain has the cofactor PLP and part of the active site, it is made up of five α helices and 6 β strands. The N-terminal domain consisting of fifteen α helices and nine β strands, is considered to be more complex and is divided in the middle of its β sheet core into subdomains. The first domain binds the effector molecule of AMP and also has a recognition site of the introconverting phosphorylase kinase and phosphatase. The second domain has the polysaccharide binding domain where phosphorylase is able to attach to the glycogen substrate. The R states of GPa and GPb are almost identical; the difference lays in the modification of the Ser-14 residue where GPa has a covalently linked phosphate group whereas GPb has a non-covalently linked sulfide group. GPa is activated by phosphorylation of the serine residue whereas GPb can be activated by the binding of AMP to the allosteric sites that are present within the molecule. GPa does not require the binding of AMP but attachment enhances the activity of the enzyme upwards to 25%.

Glycogen phosphorylase is different from other enzymes that require the cofactor PLP because instead of utilizing the pyrimidine ring, phosphorylase uses the phosphate group. The 4'aldehyde of PLP binds to the ε-amino group of lysine 680 and the 5'-phosphate of PLP has been found to be the group participating in the catalysis of glycogen phosphorylase. The binding sites in glycogen phosphorylase include: a catalytic, inhibiting, AMP, glycogen and new allosteric site. The glycogen binding site is located more than 30Å from the catalytic and allosteric sites. The residues that make up the site are Arg426, Glu433, Gly434, and Ala435. The inhibitor site binds purine analogs or fused-ring molecules such as adenosine, caffeine, FMN, NADH and AMP when there are increased concentrations available. The heterocyclic rings of the compounds bind to the inhibiting site, stablilizing it and blocking access to the catalytic center. The catalytic site can be accessed once the Ser-14 residue has been phosphorylated and conformational changes in glycogen phosphorylation have been observed. The structure and function of glycogen phosphorylase is complex, though the function of the enzyme is due to the structure. =Mechanism= In muscle, glycogen phosphorylase is activated by hormones and neural signals such as epinephrine, that stimulate phosphorylase kinase which phosphorylates the Ser-14 residue of the protein. A second messenger of cyclic AMP (cAMP) increases in concentration due to epinephrine or glucagon, and this increase results in an enzyme cascade. Activation of phosphorylase kinase is due to increased concentrations of Ca2+ or by the phosphorylation by protein kinase A which is cAMP dependent. The activated kinase in turn activates the glycogen phosphorylase enzyme by phosphorylating the Ser-14 residue. In the liver, glucagon is the primary signal which catalyzes this enzyme cascade. Glycogen phosphorylase is regulated by phosphorylation, binding of allosteric effectors and by the catalytic mechanism; phosphorylation takes glycogen phosphorylase from a disordered state to an ordered one, allosteric effector provide changes in the structure of the enzyme and when coupled with phosphorylation allow access to the buried catalytic site. The catalytic mechanism itself is dependent upon the proximity of PLP and the substrate phosphate which is directed by the surrounding groups which stabilize the interactions and create the perfect environment to phosphohydrolyze the glycosidic bond. The environment is established by the phosphate compound making a hydrogen bond with the 5'-phosphate of PLP and being stable enough to successfully cleave the bond yielding the product of glucose-1-phosphate. .

3D structures of glycogen phosphorylase
3e3n, 1pyg, 7gpb, 8gpb – rGP + AMP - rabbit

3e3o – rGP + IMP

2pyd, 2gpb – rGP + glucose

3e3l, 1c50, 1bx3, 2gpn, 1gpb – rGP

1abb – rGP + modified cofactor

1z8d – hGP + AMP + glucose - human

1fa9 – hGP + AMP

1noi, 1noj, 1nok – rGP + transition state analog + phosphate

1ygp – GP + phosphate – yeast

===Glycogen phosphorylase inhibitor complex===

3np7, 3np9, 3npa, 3msc, 3mqf, 3mrt, 3mrv, 3mrx, 3ms2, 3ms4, 3ms7, 3mt7, 3mt8, 3mt9, 3mta, 3mtb, 3mtd, 3nc4, 3l79, 3l7a, 3l7b, 3l7c, 3l7d, 3g2h, 3g2i, 3g2j, 3g2k, 3g2l, 3cut, 3cuu, 3cuv, 3cuw, 3bcs, 3bd7, 3bd8, 3bda, 2qn3, 2qn7, 2qn8, 2qn9, 2qnb, 2qlm, 2qln, 2fet, 2ff5, 2ffr, 2f3p, 2f3q, 2f3s, 2f3u, 1ww2, 1ww3, 1xkx, 1xl0, 1xl1, 1xc7, 1p4g, 1p4h, 1p4j, 1kti, 1k06, 1k08, 1hlf, 1fs4, 1ftq, 1ftw, 1fty, 1fu4, 1fu7, 1fu8, 1ggn, 2prj, 5gpb – rGP + glucopyranosyl derivative inhibitor

3g2n – rGP + acylglucosylamine

3ebo – rGP + chrysin

3ebp, 1gfz, 1c8k, 1e1y – rGP + flavopiridol

3dd1, 3dds, 3ddw – rGP + anthranilimide

2pyi – rGP + glucosyl triazoleacetamide

1gg8 – rGP + glucoside inhibitor

2pri– rGP + deoxyglucosephosphate inhibitor

3gpb, 4gpb, 6gpb – rGP + phosphorylated glucose derivative

1gpa – rGP phosphorylated

3bcr – rGP + AZT

3bcu – rGP + thymidine

2gj4 – rGP + ligand

1gpy – rGP + glucose-6-phosphate

2gm9 – rGP + thienopyrrole

1axr – rGP + azolopyridine inhibitor

2g9q, 2g9r, 2g9u, 2g9v – rGP + iminosugar

2amv – rGP + pyridine derivative inhibitor

2ieg, 2iei – rGP + quinolone derivative inhibitor

1z62, 1uzu - rGP + indirubin derivative inhibitor

3bd6 – rGP + ribofuranosyl cyanuric acid

2qrg, 2qrh, 2qrm, 2qrp, 2qrq – rGP + spiro-isoxazoline inhibitor

1a8i - rGP + spiro-hydantoine inhibitor

2qn1, 2qn2 – rGP + pentacyclic triterpene inhibitor

2off, 9gpb - rGP + allosteric inhibitor

1wv0, 1wv1, 1wuy - rGP + acyl urea derivative inhibitor

1z6p, 1z6q - rGP + AMP site inhibitor

1b4d – rGP + amidocarbamate inhibitor

1p29, 1p2b, 1p2d, 1p2g - rGP + cyclodextrin derivative inhibitor

1lwn, 1lwo – rGP + hypoglycaemic drug

2gpa, 2amv - rGP + antidiabetic drug

1h5u - rGP + antidiabetic drug + glucose

1c8l - rGP + antidiabetic drug + caffeine

2zb2 – hGP + carboxamide derivative + glucose

3ceh, 3cej, 3cem – hGP + allosteric inhibitor

2qll – hGP + GL C terminal peptide

2ati, 1wut – hGP + acyl urea derivative inhibitor

1xoi - hGP + indoloyl derivative inhibitor

1fc0 - hGP + glucopyranosyl derivative

1em6, 1exv – hGP + nucleoside + inhibitor

1l5q, 1l7x – hGP + caffeine + glucopyranosyl derivative + inhibitor

1l5r - hGP + riboflavin + glucopyranosyl derivative + inhibitor

1l5s - hGP + uric acid + glucopyranosyl derivative + inhibitor

Additional Resources
For additional information, see: Carbohydrate Metabolism

=References=